EP2700950B1 - Method for analyzing protein-protein interaction on single-molecule level within the cellular environment - Google Patents

Method for analyzing protein-protein interaction on single-molecule level within the cellular environment Download PDF

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EP2700950B1
EP2700950B1 EP12774636.0A EP12774636A EP2700950B1 EP 2700950 B1 EP2700950 B1 EP 2700950B1 EP 12774636 A EP12774636 A EP 12774636A EP 2700950 B1 EP2700950 B1 EP 2700950B1
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proteins
substrate
cell
protein
bound
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EP2700950A4 (en
EP2700950A2 (en
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Tae-Young Yoon
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Korea Advanced Institute of Science and Technology KAIST
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the present invention relates to a method of analyzing protein-protein interactions, and more specifically, to a method of analyzing protein-protein interactions, which can analyze the protein-protein interactions at a single molecular level in the actual intracellular environment, and also can compare and confirm each cell state and activation levels of first proteins by comparing after varying of a type of cell supplying the first proteins for observing interactions between the first proteins and the second proteins.
  • a cell maintains a life phenomenon by performing several biological functions, such as gene expression, cell growth, cell cycle, metabolism, signal transduction, and the like, through various and complex protein-protein interactions. Accordingly, the understandings of intracellular protein-protein interactions and functions of the interactions have been the foundation of the understandings of the life phenomenon and are an essential part for developing new drugs and treating diseases.
  • a representative method of investigating protein-protein interactions in vitro is an affinity chromatography method.
  • a method of investigating the protein-protein interactions analyzes the interactions in the isolation of proteins from other intracellular materials by purifying each of the proteins that exist in the cell for analyzing the protein-protein interactions. Accordingly, there is a limit to analyze the protein-protein interactions at the single molecular level in the actual intracellular environment with co-existing other proteins, and the like.
  • a method of investigating the protein-protein interactions according to the conventional technologies has a problem that the degrees of effects of other proteins on specific protein-protein interactions cannot be analyzed when other proteins are involved with the interactions in the actual intracellular environment.
  • US 2003/186311 A1 discloses methods for detecting molecular interactions comprising arrays (e.g., glass slides coated with PEG on which antibodies are covalently attached). The detection is performed using near field scanning probe microscopy which enables detection at the single molecular level.
  • WO 2009/100442 A2 discloses a nanofluidic device and method for determining molecule-molecule interactions with a single molecule detection system comprising contacting a sample (e.g., breast cancer cell lysate) with a binding moiety (such as an antibody), contacting the binding moiety with a detection moiety, introducing the sample into a microchannel for single fluorescence signal detection, and detecting the complex.
  • a sample e.g., breast cancer cell lysate
  • a binding moiety such as an antibody
  • Sako Y. et al., Nat. Cell Biol., 2000 (2): 168-172 discloses a method and apparatus for single molecule imaging of the EGFR signalling pathway, and in particular of activated EGFR.
  • the detection is performed with total internal reflection (TIR) fluorescence microscopy.
  • TIR total internal reflection
  • Wabuyele M.B. et al., Meth. Mol. Biol., 2005 (300): 437-452 discloses the advantages and usefulness of near-field scanning optical microscopy (NSOM) for imaging biological molecules especially for investigating the colocalization of proteins and for the bioanalysis at nanometer resolution.
  • NSOM near-field scanning optical microscopy
  • US 2007/128665 A1 discloses a method for analysing interactions between two proteins such as small GTPases (exemplified with the interaction between ras and raf) and for identifying a compound capable of inhibiting such interaction. This is realized by following GTP loading on a membrane bound small GTPase (e.g., ras) and following the interaction ras-raf by fluorescence microscopy, especially translocation from the membrane to the cytosol after activation, using a GFP-raf.
  • a membrane bound small GTPase e.g., ras
  • fluorescence microscopy especially translocation from the membrane to the cytosol after activation, using a GFP-raf.
  • an object of the present invention is to provide a method of analyzing protein-protein interactions, which can analyze the protein-protein interactions at a single molecular level in the actual intracellular environment, as well as can compare and confirm each cell state and activation levels of first proteins by comparing after varying of a type of cells supplying the first protein for observing interactions between the first proteins and the second proteins.
  • the present disclosure describes a method of measuring activated protein concentrations in a cell lysate, which can compare and measure quantitatively specific activated protein concentrations in the cell of an experimental group and the cell of a control group.
  • a method of analyzing interactions between first proteins and second proteins by at a single molecular level comprising: (a) preparing at least two substrates, in which first protein-binding molecules that are biomolecules to be bound to the first proteins are attached to each of the substrates; (b) inducing binding between the first proteins and the first protein-binding molecules on the first substrate and the second substrate, respectively, by supplying the first proteins included in the control group-cell to the first substrate among the two substrates and supplying the first proteins included in the experimental group-cell to the second substrate among the two substrates, wherein the concentration of the control group cell should be equal to the concentration of the experimental group cell; (c) supplying cell lysates of cells including the marker-tagged second proteins to the first substrate and the second substrate, respectively, when the first proteins and the first protein-binding molecules are bound to the first substrate and the second substrate, respectively, wherein the concentration of the second proteins in the cytoplasmic cell lysates to
  • control group-cell is a normal cell and the experimental group-cell is a tumor cell.
  • the step (d) includes measuring a fluorescent signal having a specific wavelength generated by the markers tagged to the second proteins bound to the first proteins using an optical apparatus generating a near-field.
  • the fluorescent signal having the specific wavelength is cumulatively measured for a predetermined time period.
  • the fluorescent signal having the specific wavelength is measured in real-time under the presence of the cell lysates supplied to the substrate.
  • the step (b) includes supplying the cell lysates of the control group-cell to the first substrate and supplying the cell lysates of the experimental group-cell to the second substrate.
  • the method further comprises supplying a buffer solution to the substrate before the step (c).
  • the present disclosure further describes a method of measuring activated protein concentration in a cell lysate, comprising: (a) preparing a substrate attaching first protein-binding molecules that are biomolecules to be bound with first proteins; (b) inducing binding of the first proteins and the first protein-binding molecules by supplying the cell lysates including the first proteins to the substrate; (c) supplying the cell lysates including the marker-tagged second proteins when the first proteins are bound to the first protein-binding molecules on the substrate; and (d) analyzing interactions between the first proteins and the second proteins on the substrate.
  • the method further comprises (e) repeating the steps (b) to (d) by increasing the concentration of the cell lysates including the first proteins in the step (b), by a predetermined ratio.
  • the step (d) includes measuring a generation frequency of a fluorescent signal having a specific wavelength generated by the markers tagged to the second proteins bound to the first proteins in any configured observation region on the substrate.
  • the first proteins that interact with the second proteins in the step (d) are activated first proteins among the first proteins bound to the first protein-binding molecules.
  • the step (d) includes measuring a fluorescent signal having a specific wavelength generated by the markers tagged to the second proteins bound to the first proteins using an optical apparatus generating a near-field.
  • the present disclosure further describes a method of measuring activated protein concentration in a cell lysate, comprising: (a) preparing at least two substrates, to which first protein-binding molecules that are biomolecules to be bound to first proteins are attached, respectively; (b) inducing binding between the first proteins and the first protein-binding molecules on the first substrate and the second substrate, respectively, by supplying the first proteins included in the control group-cell to the first substrate among the two substrates and supplying the first proteins included in the experimental group-cell to the second substrate among the two substrates; (c) supplying cell lysates including the marker-tagged second proteins to the first substrate and the second substrate, respectively, when the first proteins and the first protein-binding molecules are bound to the first substrate and the second substrate, respectively; and (d) comparing and analyzing the interactions between the first proteins and the second proteins on the first substrate and the second substrate in the supply of the cell lysates to the first substrate and the second substrate, respectively.
  • the method further comprises (e) repeating the steps (b) to (d) while increasing the concentrations of the control group-cell lysate and the experimental group-cell lysate in the step (b), by a predetermined ratio.
  • the step (d) includes comparing and measuring a generation frequency of a fluorescent signal having a specific wavelength generated by the markers tagged to the second proteins bound to the first proteins in any configured observation region on the substrate.
  • the first proteins that interact with the second proteins in the step (d) are activated first proteins among the first proteins bound to the first protein-binding molecules.
  • the step (d) includes measuring a fluorescent signal having a specific wavelength generated by the markers tagged to the second proteins bound to the first proteins using an optical apparatus generating a near-field.
  • the present invention it is possible to analyze the protein-protein interactions at a single molecular level in the actual intracellular environment, as well as to compare and confirm each cell state and activation levels of first proteins by comparing after varying of a type of cells supplying the first protein for observing interactions between the first proteins and the second proteins.
  • the present disclosure further describes a method of measuring activated protein concentrations in a cell lysate, which can compare and measure quantitatively specific activated protein concentrations in the cell of an experimental group and the cell of a control group.
  • Fig. 1 is a flowchart illustrating a procedure of a method of analyzing protein-protein interactions according to an embodiment of the present invention.
  • an analyst attaches first protein-binding antibodies (Primary antibody) that are antibodies to be bound to the first proteins to a substrate that is a quartz slide coated with polyethylene glycol (PEG), in order to analyze the interactions between the first proteins and the second proteins that are two specific proteins at a single molecular level (S110).
  • first protein-binding antibodies Primary antibody
  • PEG polyethylene glycol
  • the first proteins are h-Ras proteins and the second proteins are Ras-binding domain (RBD) proteins of C-Raf.
  • RBD Ras-binding domain
  • the antibodies to be bound to the first proteins may include biomolecules to be bound to the first proteins, such as DNA, RNA, or liposomes having specific components to be bound to proteins, and the like, in addition to antibodies.
  • the analyst induces bindings between the first proteins and the first protein-binding antibodies by supplying (S120) cytoplasmic lysate of the cell including the first proteins to the substrate (S130).
  • cell lysates such as cytolysate, diluted cytoplasmic lysate, diluted or purified cytolysate, and the like, may be used in addition to the cytoplasmic lysate.
  • the analyst may pre-treat for an expression of predetermined first fluorescent proteins, such as m-Cherry, and the like, to the first proteins.
  • first fluorescent proteins such as m-Cherry, and the like
  • the analyst may confirm whether the first proteins are bound to a plurality of first protein-binding antibodies attached on the substrate from the change of wavelength by the first fluorescent proteins that are expressed to the first proteins (i.e., measuring individual single molecule signal generated from the first fluorescent proteins) by performing an observation of the surface of the substrate using a total internal reflection fluorescence microscope.
  • the first fluorescent proteins when expressed in the first proteins, whether or not the first proteins are bound to the antibodies may be confirmed through the total internal reflection fluorescence microscope, and thus the number of the first proteins bound to the antibodies attached on the substrate and binding density thereof may be accurately measured.
  • the analyst removes the remaining materials included in the cytoplasmic lysate except the first proteins from the substrate by supplying a buffer solution to the substrate (S140).
  • the analyst manipulates for an expression of second fluorescent proteins through a genetic manipulation of second proteins being in different relevant cells among the same cells as the above-described cells (S150).
  • the second fluorescent molecules may be attached or connected to the second proteins by a physical-chemical method.
  • the above described step, S150 may be performed in advance before the above-described step, S110 in order to smoothly progress the analysis.
  • the second fluorescent proteins preferably have wavelength range different from the first fluorescent proteins, when the first fluorescent proteins are m-Cherry proteins, the second fluorescent proteins may be eGFPs (enhanced Green Fluorescent Protein) that is a green fluorescent protein.
  • the analyst supplies the whole cytoplasmic lysate of the cell having the expressed second fluorescent proteins inside the second proteins from the above-described step, S150, to the substrate (S160).
  • At least part of the plurality of the first protein-binding antibodies attached on the surface of the substrate is bound to each first protein.
  • the first proteins (Cellular Ras) on the surface of the substrate interact with the second proteins in the same condition as the intracellular environment in the coexisting with the second proteins (eGFP-cRBD) included in the cytoplasmic lysate and native proteins in the whole cell lysate.
  • each first protein (Cellular Ras) bound to each antibody (Anti-Ras Primary antibody) attached on the surface of the substrate interacts with the second protein (eGFP-cRBD) in the same condition as the intracellular environment at the single molecular level thereby repeating binding and the unbinding.
  • cell lysates such as cytolysate, diluted cytoplasmic lysate, diluted or purified cytolysate, and the like, may be used in addition to the cytoplasmic lysate.
  • the analyst can confirm that the wavelength on the surface of the substrate is changed from 473 nm to 520 nm by eGFP that is a fluorescent protein that is expressed to the second proteins through performing an observation of substrate surface using the total internal reflection fluorescence microscope having 473 nm wavelength.
  • the binding state between the first proteins and the second proteins can be confirmed through a detection of fluorescence signals having a specific wavelength bandwidth (520 nm) generated from the second fluorescent protein (eGFP) located on the surface of the substrate through the binding between the first proteins and the second proteins, and the analyst can analyze the interactions, such as the frequencies of binding and unbinding between the first proteins and the second proteins, and the like, at the single molecular level by continuously observing the change of wavelength at each of antibodies attached on the surface of the substrate (S170).
  • eGFP second fluorescent protein
  • the interactions such as the frequencies of the binding and unbinding between the first proteins and the second proteins, and the like, can be analyzed in the same environment as the intracellular environment by measuring the fluorescence signals having a specific wavelength in real time under the presence of the cytoplamic lysate supplied to the substrate in the above-described step, S160.
  • the analyst may compare and analyze the interactions between the first proteins and the second proteins in the cell of a normal state (cell of a control group) and the interactions between the first proteins and the second proteins in cell of an abnormal state (cell of an experimental group).
  • a normal breast tissue cell is changed to a tumor cell as shown in Fig. 4 by artificially maximizing an activation of Ras protein in the cell isolated from a breast tissue of a human body, and the normal breast tissue cell was used as the cell of a control group and the breast tissue cell to be changed to the tumor cell was used as the cell of an experimental group.
  • Fig. 5 is a flowchart illustrating a procedure of a method of analyzing protein-protein interactions according another embodiment of the present invention.
  • the analyst prepares two quartz slide substrates with the same size coated with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the analyst attaches the first protein-binding antibodies (Anti-Ras Primary antibody) that are the antibodies to be bound to the first proteins on both of two substrates (first substrate and second substrate) as shown in Fig. 2 (S210).
  • Anti-Ras Primary antibody the first protein-binding antibodies
  • the antibody to be bound to the first proteins may include biomolecules to be bound to the first proteins, such as DNA, RNA, or liposomes having specific components to be bound to proteins, and the like, in addition to antibodies.
  • the analyst induces binding between the first proteins and the first protein-binding antibodies (Anti-Ras Primary antibody) on each of the first substrate and the second substrate by supplying (S220) the cytoplasmic lysate of the cell of the control group including the first proteins to the first substrate and the cytoplasmic lysate of the cell of the experimental group including the first proteins to the second substrate (S230).
  • Anti-Ras Primary antibody the first protein-binding antibodies
  • the concentration of the first proteins included in the cell of the control group should be equal to the concentration of the first proteins included in the cell of the experimental group. To achieve this, it should be confirmed whether both of the concentrations of the first proteins included in the cells of the control group and the experimental group are one and the same by measuring both of the concentrations. When both of the concentrations are not the same as each other, the above-described step, S230, should be performed after both of the concentrations are adjusted to be the same as each other through diluting, and the like.
  • cell lysates such as cytolysate, diluted cytoplasmic lysate, diluted or purified cytolysate, and the like, may be used in addition to the cytoplasmic lysate.
  • the analyst may pre-treat for an expression of predetermined first fluorescent proteins, such as m-Cherry, and the like, to the first proteins.
  • first fluorescent proteins such as m-Cherry, and the like
  • the analyst may confirm whether the first proteins are bound to the plurality of the first protein-binding antibodies attached on the substrate from the change of wavelength by the first fluorescent proteins that are expressed in the first proteins (i.e., measuring single molecule signal generated from the first fluorescent proteins) by performing an observation of the surface of the substrate using the total internal reflection fluorescence microscope.
  • the first fluorescent proteins when expressed in the first proteins, whether or not the first proteins are bound to the antibodies may be confirmed through the total internal reflection fluorescence microscope, and thus the number of the first proteins bound to the antibodies attached on the substrate and binding density thereof may be accurately measured.
  • the analyst removes the remaining materials included in the cytoplasmic lysate except the first proteins from the substrates by supplying a buffer solution to the substrates (S240).
  • the analyst manipulates to express second fluorescent proteins through a genetic manipulation of second proteins that exist in specific cells (S250).
  • the second fluorescent proteins may be attached or connected to the second proteins by a physical-chemical method.
  • the second fluorescent proteins may preferably have a wavelength region different from that of the first fluorescent proteins, and thus when the first fluorescent proteins are m-Cherry proteins, the second fluorescent proteins may be eGFP (enhanced Green Fluorescent Protein) that is a green fluorescent protein.
  • eGFP enhanced Green Fluorescent Protein
  • the analyst supplies the whole cytoplasmic lysates of the cell with the expressed second fluorescent proteins inside the second proteins in the above-described step, S260, to the first substrate and the second substrate, respectively (S260).
  • the concentration of the second proteins in the cytoplasmic lysates to be supplied to the first substrate and the second substrate, respectively, should be the same as each other. To achieve this, it should be confirmed whether both of the concentrations of the second proteins included in two cytoplasmic lysates are one and the same by measuring both of the concentrations. When both of the concentrations are not the same as each other, the above-described step, S260, should be performed after both of the concentrations are adjusted to be the same as each other through diluting, and the like.
  • cell lysates such as cytolysate, diluted cytoplasmic lysate, diluted or purified cytolysate, and the like, may be used in addition to the cytoplasmic lysate.
  • the first proteins of the cell of the control group and the first proteins of the cell of the experimental group are bound to the plurality of the first protein-binding antibodies that are attached to the surfaces of the first substrate and the second substrate, respectively.
  • the first proteins on each surface of the substrates interact with the second proteins in the same state as the intracellular environment that co-exists with the second proteins (eGFP-cRBD) included in the cytoplasmic lysate and native proteins in the whole cell lysate.
  • the analyst may confirm binding state of the first proteins and the second proteins through a detection of the fluorescent signal having a specific wavelength bandwidth (520 nm) generated from the second fluorescent protein (eGFP) to be located on the surface of the substrate through binding between the first proteins and the second proteins, and may compare and analyze the interactions, such as the frequencies of binding and unbinding between the first proteins and the second proteins on the first substrate and the second substrate, and the like, by continuously observing the change of wavelength of each antibody attached on the surface of the substrate (S270).
  • eGFP specific wavelength bandwidth
  • Fig. 6 is a graph showing the signal observed on the first substrate bound with the first proteins in the cell of the control group and Fig. 7 is a graph showing the signal observed on the second substrate bound with the first proteins in the cell of the experimental group.
  • the first proteins interacting with the second proteins may become to be the activated first proteins, and thus the activation levels of the first proteins in the cell of a control group and the first proteins in the cell of a experimental group may be quantitatively measured by comparing the interactions between the first proteins and the second proteins in the cell of the control group and the interactions between the first proteins and the second proteins in the cell of the experimental group as mentioned above.
  • Fig. 8 is a diagram illustrating a method of comparing the activated protein concentrations in the cells of a control group and an experimental group according to another embodiment of the present invention.
  • the analyst prepares two quartz slide substrates with the same size coated with polyethylene glycol (PEG) and attaches the first protein-binding antibodies (Anti-Ras Primary antibody) that are antibodies to be bound to each first protein to two substrates (first substrate and second substrate) as shown in Fig. 2 (S310).
  • PEG polyethylene glycol
  • the analyst induces binding between the first proteins and the first protein-binding antibodies (Anti-Ras Primary antibody) on each of the first substrate and the second substrate, respectively, by supplying (S320) the cell lysates of the control group-cell including the first proteins to the first substrate and supplying (S330) the cell lysates of the experimental group-cell including the first proteins to the second substrate.
  • first protein-binding antibodies Anti-Ras Primary antibody
  • the concentration of the first proteins included in the cell of the control group should be equal to the concentration of the first proteins included in the cell of the experimental group. To achieve this, it should be confirmed whether both of the concentrations of the first proteins included in the cells of the control group and the experimental group are one and the same by measuring both of the concentrations.
  • the analyst may pre-treat for an expression of predetermined first fluorescent proteins, such as m-Cherry, and the like, to the first proteins.
  • first fluorescent proteins such as m-Cherry, and the like
  • the analyst may confirm whether the first proteins are bound to the plurality of the first protein-binding antibodies attached on the substrate from the change of wavelength by the first fluorescent proteins that are expressed to the first proteins (i.e., measuring single molecule signal generated from the first fluorescent proteins) by performing an observation of the surface of the substrate using the total internal reflection fluorescence microscope.
  • the first fluorescent proteins when expressed in the first proteins, whether the first proteins are bound to the antibodies may be confirmed through the total internal reflection fluorescence microscope, and thus the number of the first proteins bound to the antibodies attached on the substrate and binding density thereof may be accurately measured.
  • the analyst removes the remaining materials included in the cytoplasmic lysate except the first proteins from the substrates by supplying a buffer solution to the substrates.
  • the analyst manipulates to express second fluorescent proteins through a genetic manipulation of second proteins that exist for specific cells.
  • the second fluorescent proteins may be attached or connected to the second proteins by a physical-chemical method.
  • the second fluorescent proteins may preferably have a wavelength region different from that of the first fluorescent proteins, and thus when the first fluorescent proteins are m-Cherry proteins, the second fluorescent proteins may be eGFP (enhanced Green Fluorescent Protein) that is a green fluorescent protein.
  • eGFP enhanced Green Fluorescent Protein
  • the analyst supplies the whole cell lysate of the cell with the expressed second fluorescent proteins inside the second proteins to the first substrate and the second substrate, respectively (S340).
  • the concentration of the second proteins in the cell lysate to be supplied to the first substrate and the second substrate, respectively should be the same as each other. To achieve this, it should be confirmed whether both of the concentrations of the second proteins included in two cytoplasmic lysates are one and the same by measuring both of the concentrations. When both of the concentrations are not the same as each other, the above-described step, S340, should be performed after both of the concentrations are adjusted to be the same as each other through diluting, and the like.
  • the first proteins of the cell of the control group and the first proteins of the cell of the experimental group are bound to the plurality of the first protein-binding antibodies that are attached to the surfaces of the first substrate and the second substrate, respectively.
  • the whole cell lysates such as the cytoplasmic lysate including the second proteins on the above-mentioned surface of the substrate, and the like, are supplied as shown in Fig. 3
  • the first proteins on each surface of the substrates interact with the second proteins in the same state as the intracellular environment that allows for the coexistence of the second proteins (eGFP-cRBD) included in the cytoplasmic lysate and native proteins in the whole cell lysate.
  • the analyst may confirm binding state of the first proteins and the second proteins through a detection of the fluorescent signal having a specific wavelength bandwidth (520 nm) generated from the second fluorescent protein (eGFP) to be located on the surface of the substrate through binding between the first proteins and the second proteins, and may compare and analyze the interactions, such as the frequencies of binding and unbinding between the first proteins and the second proteins on the first substrate and the second substrate, and the like, by continuously observing the change of wavelength to each antibody attached on the surface of the substrate (S350).
  • a specific wavelength bandwidth 520 nm
  • eGFP second fluorescent protein
  • the analyst repeats the above-described steps, S320 to S350, while gradually and equally increasing the concentration of the first proteins included in the cell lysate of the control group and the concentration of the first proteins included in the cell of the experimental group supplied to the first substrate and the second substrate in the above-described steps, S320 and S330.
  • Fig. 9 is a diagram showing the experimental results of measuring the interaction between the first proteins and the second proteins with gradually increasing the concentration of the first proteins included in the cell of the experimental group.
  • the cell lysate of the cell of the experimental group including the first proteins (HRas) of 1 nM concentration is supplied to the second substrate to induce binding between the first proteins and the first protein-binding molecules and then the generated signal of the fluorescent signal having a specific wavelength generated by markers tagged to the second proteins is measured by using an optical apparatus generating a near-field in the supply of the cell lysates including the marker-tagged second proteins to the second substrate.
  • the optical apparatus can measure a frequency of fluorescent signal generation with each ROI (Region of Interest) as the center after setting ROI of any 1.1 ⁇ m 2 size with the point of sensing the fluorescent signal having a specific wavelength that exceeds a predetermined threshold as the center.
  • ROI Region of Interest
  • the cell lysate of the cell of the experimental group including the first proteins (HRas) of 2 nM concentration is supplied to the second substrate to induce binding between the first proteins and the first protein-binding molecules and then the generated signal of the fluorescent signal with a specific wavelength generated by a marker tagged to the second proteins is measured by using an optical apparatus generating a near-field in the supply of the cell lysate including the marker-tagged second proteins to the second substrate.
  • the frequency of the fluorescent signal generation averagely measured in eight ROI in Fig. 9(b) is equal to that of Fig. 9(a) .
  • the average value of the frequency of the fluorescent signal generation becomes to increase in each ROI and then the average value of the frequency of the fluorescent signal generation is continuously increased with an increasing concentration of the experimental group-cell lysate, continuously.
  • the threshold of the experimental group-cell lysate of the tumor cell including the first proteins (HRas), in which the average value of the frequency of the fluorescent signal generation is started to increase in each ROI with the size of 1.1 ⁇ m 2 is measured to be 5 nM.
  • the cell lysate of the experimental group-cell including the first proteins (HRas) with 3 nM concentration is supplied to the second substrate to induce binding between the first proteins and the first protein-binding molecules. From that point, when the frequency of the fluorescent signal generation with specific wavelength generated by the markers tagged to the second proteins is measured by using an optical apparatus in a plurality of ROIs as its average value in the supply of the cell lysate including the marker-tagged second proteins to the second substrate, the frequency of the fluorescent signal generation is just 8 times in 30 seconds.
  • the cell lysate of the experimental group-cell including the first proteins (HRas) with 41.3 nM concentration that exceeds a threshold concentration (5 nM) is supplied to the second substrate to induce binding between the first proteins and the first protein-binding molecules.
  • a threshold concentration 5 nM
  • the average value of the frequency of the fluorescent signal in a plurality of ROIs is measured.
  • Fig. 14 it has been seen that it is constantly maintained at 5 nM that is a threshold concentration value, and then increased after exceeding the threshold concentration value.
  • the analyst may calculate the ratio of activated first proteins among the first proteins included in the cell lysate of the experimental group-cell.
  • the analyst may quantitatively calculate the ratio of activated first protein concentration included in the cell of the control group and the activated first protein concentration included in the cell of the experimental group by comparing the threshold concentration values in the control group-cell and the experimental group-cell by repeating the experiments as mentioned above in the control group-cell.
  • the analyst measures the threshold concentration value in the control group-cell under the same condition while slowly increasing the concentration of the cell lysate of the control group-cell including the first proteins (HRas) along with the experimental procedure as mentioned above, or before and after the experimental procedure to the experimental group-cell in order to achieve the threshold concentration value in the experimental group-cell.
  • HRas first proteins
  • the analyst induces binding between the first proteins and the first protein-binding molecules by supplying the cell lysate of the control group-cell including the first proteins (HRas) with 3.5 nM concentration to the second substrate. From that point, when the frequency of the fluorescent signal generation having a specific wavelength generated by the markers tagged to the second proteins is measured by using an optical apparatus generating a near-field in a plurality of ROIs as its average value in the supply of the cell lysate including the marker-tagged second proteins to the second substrate, the frequency of the fluorescent signal generation is just 6 times in 30 seconds.
  • the frequency of the fluorescent signal generation measured in a plurality of ROIs as its average value is constant at 6 times in 30 seconds.
  • the cell lysate of the control group-cell including the first proteins (HRas) with 49 nM concentration is supplied to the second substrate to induce binding between the first proteins and the first protein-binding molecules.
  • the frequency of the fluorescent signal generation with a specific wavelength generated by a marker tagged to the second proteins is measured by using an optical apparatus generating a near-field in a plurality of ROIs as its average value in the supply of the cell lysate including the marker-tagged second proteins to the second substrate, the frequency of the fluorescent signal generation is measured to be 7 times in 30 seconds, that is increased.
  • the analyst can confirm the fact that the activated first proteins included in the experimental group-cell that is a tumor cell are 10 times larger than those of the activated first proteins included in the control cell that is a normal cell based on the fact that the threshold concentration value in the experimental group-cell is measured to be 5 nM and the threshold concentration value in the control group-cell is measured to be 50 nM
  • each cell state and activation levels of first proteins can be compared and confirmed by comparing after varying of a type of cells supplying the first proteins for observing interactions between the first proteins and the second proteins.
  • the protein-protein interactions can be analyzed at a single molecular level in the actual intracellular environment.
  • specific activated protein concentrations can be quantitatively measured in the cell of an experimental group and the cell of a control group.
  • the present invention can be used in medical industry.

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